Vaccine manufacture for active immunization containing hepatitis B surface antigen and associated antigen

ABSTRACT

A process for preparing a vaccine containing unprecipitated filaments and hepatitis B Dane particle specific antigens by removal from a blood serum of other proteinaceous matter such that the serum contains less than 10% proteinaceous matter other than that associated with hepatitis B surface antigen or the filament or Dane particle specific antigen. Any virus present is inactivated, and the antigenous mass is diluted with a physiologically acceptable medium.

ACKNOWLEDGEMENT OF H.E.W. SUPPORT

The invention described herein was made in the course of or under agrant from the National Institute of Health, Department of Health,Education and Welfare.

This is a division of application Ser. No. 631,961, filed Nov. 17, 1975.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to an effective vaccine against viral hepatitiswhich is characterized by containing large particles of filamentousand/or Dane particles characterized by a particle size of 20 to 50nanometers (nm) said vaccine being further characterized by thesubstantial absence of antibodies to the antigens therein, the vaccinehaving both hepatitis B surface antigens as well as antigens which arespecific for the virion itself such as e-antigen(s). The invention isalso directed to the preparation of such vaccine, its purification andthe multiplication of the vaccine in a host animal and the use thereofin active immunization against various forms of viral hepatitis.

2. Discussion of the Prior Art

Epidemiologic and human volunteer studies carried out between 1939 and1967 prove the existence of two major hepatitis viruses -- virus A andvirus B. It became increasingly clear that further understanding of thevirology of these viruses and the prevention of the diseases require thedevelopment of immunologic methodology. Considerable research focused onthese viruses, particularly that known as virus B. Virus B wasassociated with those patients who had undergone a number of injectionsor transfusion of blood and, hence, the virus became known as serumhepatitis. As a result of the discovery of antigens specific forhepatitis B virus infections [Prince et al., Am. J. Hyg. 79, 365, 1964;Blumberg et al., JAMA 191, 541, 1965; Prince, Proc. Nat. Acad. Sci. USA,60, 814, 1968] it became apparent that "serum hepatitis virus" was, infact, contagious under certain circumstances. It therefore becameappropriate to alter the terminology. The Committee on Viral Hepatitisof the Division of Medical Sciences, Academy of Sciences, NationalResearch Council therefore recommended in 1972 a return to the formerterminology, namely hepatitis A virus for infectious hepatitis virus anda hepatitis B virus for serum hepatitis virus. At the same time thecommittee recommended the use of the term hepatitis B antigen (HB_(s)Ag) for the antigen in place of the previous term, e.g., Australiaantigen (Au Ag), which had been employed inasmuch as the antigen hadfirst been discovered in the blood of Australian aboriginies; SerumHepatitis Antigen (SH); Hepatitis Associated Antigen (HAA); etc.

Subsequently, studies focused upon the antigenic specificities ofhepatitis B virus infections and reseach centered about sub-typeantigens now termed HB_(s) Ag/adywr, etc., the hepatitis B core antigen(HB_(c) Ag) discovered by Almeida, the e-antigen(s) discovered byMagnius and Espmark as well as hepatitis B surface antigen (HB_(s) Ag).Antibody to Hb_(s) Ag is termed Anti-HB_(s).

Many of the early researchers, including Blumberg, were of the beliefthat hepatitis B antigens were of host genetic origin. Otherresearchers, including Prince and Vnek, considered that hepatitis Bantigens were determined by the genome of hepatitis B virus. It becameincreasingly evident that active immunization would be required toprevent infections by this virus, and to that end research focused uponthe preparation of a vaccine.

Heretofore, particles in human sera having a particle size in the rangeof about 20 to 25 nm were regarded as the predominant particles whichcarry the hepatitis B surface antigens. Researchers focused about theseparation of these particles from the other proteinaceous material inthe blood, and especially from the larger 42 nm Dane particle which manysuspected to be the infective virion. It was postulated that if theantigen could be purified to the required extent, and Dane particlesremoved, there could be provided a material which when diluted with aphysiologically acceptable medium and further inactivated for safety,would provide a vaccine for active immunization.. To this end Blumbergin U.S. Pat. No. 3,636,191 recommended the treatment of blood plasmafrom HB_(s) Ag carriers with a mixture of enzymes to digest newparticle-associated proteins, followed by conventional centrifugationsteps to recover purified hepatitis B surface antigen on particleshaving a particle size of about 22 nm. Blumberg et al. had recognizedthat the 22 nm hepatitis B surface antigen-associated particles were inthe form of a shell which had no core and which was substantiallyresistant to the various enzymes employed in the digestion procedure.Blumberg provided a vaccine having a density of the order of 1.21 Gm/ccwhich, according to Blumberg, can be diluted with a physiologicallyacceptable medium and employed as an active immunizing agent.

The vaccine provided by Blumberg may be effective in inducing synthesisof antibodies in a host animal such as a human being, however, suchantibodies are now known by us to be of limited specificity and do notinclude antibodies to the unique specificity(s), e.g., e-antigen,present only on the larger particles, such as the Dane particle (i.e.,the virion). The vaccine proposed by Blumberg is thus not protectiveagainst the large doses of virus to which people are frequently exposed,e.g., in transfusions. (See FIGS. 1 and 2 which show the theoreticalbasis for this conclusion.) While it would theoretically be possible toproduce very high titers of anti-HBs no acceptable means for doing thisin man has been provided. Theoretically one could employ Freund'sadjuvant to stimulate production of high quantities of antibodies. Thenthe very high concentrations of anti-HBs might effectively neutralizeboth hepatitis B surface antigen and the e-antigen-associated virions(see FIG. 2). However, because of potential carcinogenicity, and otheradverse side effects of Freund's and other known adjuvants, no method ispresently known for the production of such high titers of anti-HBs inman.

It has therefore become desirable to provide a vaccine against viralhepatitis which not only stimulates and induces the production ofanti-HBs, but also that of antibodies specific to the virion itself,i.e., the 42 nm Dane particle, such as e-antigen(s). Production of sucha vaccine has previously been impossible when pools of human HB_(s)Ag-containing plasma are used as source material. The reason for this isthat such plasma pools contain an excess of anti-e antibody. Thiscombines with any e-antigen-containing particles, resulting in theirprecipitation. The precipitated e-antigen-containing particles aredifficult if not impossible to purify by conventional methods, andtherefore do not appear in the final product.

It is therefore an object of this invention to provide a means forisolating hepatitis B surface antigen together with free uncombined andunprecipitated e-antigen-containing particles to provide a vaccine foruse in active immunization. It is a further object of this invention toprovide a means for the multiplication of such antigens in a hostanimal, and followed by purification of the HB-associated antigenicmaterial from the host animals blood to produce a broad and effectivevaccine against viral hepatitis and related disorders.

SUMMARY OF THE INVENTION

In accordance with this invention there is provided a vaccine againstviral hepatitis comprising:

A. antigenic particles having a particle size in the range of 20 to 50nm, said antigenic particles containing unprecipitated free uncombinedhepatitis B surface antigens;

B. said vaccine having less than 10 units of antibodies of hepatitis Bsurface antigen per 1,000 units of hepatitis B surface antigen;

C. at lease 5% of the particles of said vaccine in the size range of 30to 50 nm containing unprecipitated e-antigen(s), or similar Daneparticle specific antigens. Such antigens are hereinafter loosely termed"e-antigen" as a conventional designation for what might more properlybe called HB_(D) Ag (Hepatitis B Dane particle specific antigens);

D. said hepatitis B antigen and said e-antigens being present in saidvaccine in an amount sufficient to produce antibodies when introducedinto a host animal, the balance being a medium which is physiologicallyacceptable.

Surprisingly, it has been found that a "select" few (Ca 2%) ofclinically well people who carry hepatitis B surface antigen haveparticles containing free e-antigen. By this is meant that their bloodnot only contains HB_(s) Ag in a shell of particle size of about 22 nm(as reported by Blumberg and others) but also contains larger particleswhich are either filamentous particles with a diameter of 20 to 45 nm orDane particles of generally cylindrical configuration having an averagediameter of 42 nm which contain, in addition to conventional HB_(s) Ag,e-antigen(s).

It has been found that vaccine can only be produced from the blood ofthese "select" (i.e., e-antigen positive, anti-e antibody negative)donors.

An important basis for this discovery is that the fact that Daneparticles and the larger filamentous particles contain the e-antigen.This finding provides a rational basis for the associations summarizedin Tables I and II.

Additionally, it has been found that e-antigen is also present in serumin a "soluble" form having a molecular weight of less than 1,000,000generally 50,000 to 200,000, in addition to that present on the largerHB_(s) Ag-associated particles. The blood of such e-antigen positivecarriers of course, also contains the shell-like HB_(s) Ag-associatedparticles of size of about 22 nm which lack e-antigen (see FIG. 1).

The rationale, as well as the theoretical advantage of this presentvaccine, is illustrated by the scheme shown in FIG. 2. Note thatmoderate titers of anti-HB_(s) as would be provided by conventional 20nm particle vaccines, such as Blumbergs, will provide protection only ifthe challenge dose is exceedingly low; when larger quantities of HBvirus, and its associated HB_(s) Ag-containing particles, areencountered, these will neutralize small quantities of anti-HB_(s),leaving free and infective Dane particles able to infect the liver ofthe host. In marked contrast, when an anti-e response is induced thisantibody(s) can specifically combine with the infective virions (Daneparticles) without being neutralized by the vast excess (usually 500small particles per 1 Dane particle) of small HB_(s) Ag-associatedparticles.

The vaccine provided by the present invention is therefore characterizedby the presence of particles in the range of 30 to 50 nm and preferablyin the range of 35 to 45 nm. These larger particles, which are generallypresent together with particles of size of 16 to 22 nm, which alsocontain HB_(s) Ag, are generally of two major types: i.e., filaments andDane particles as described above.

The vaccine thus contains both antigenic specificities, proteins, andnucleic acids not present in the smaller HB_(s) Ag-containing particlesdescribed in U.S. Pat. No. 3,636,191 (see FIG. 1). Note that Daneparticles have a core as well as a shell, the core containing DNA, DNApolymerase and HB_(c) Ag. The shell of the Dane particle contains HB_(s)Ag as well as e-antigen(s) and similar Dane-specific antigens.Therefore, the overall vaccine must contain particles having a higherdensity than the vaccine particles of U.S. Pat. No. 3,636,191. Thedensity of the vaccine of the present invention is in the range of 1.23to 1.34 preferably 1.24 to 1.34 as determined by isopycnic densitygradient centrifugation in cesium chloride density gradients.

The vaccine is characterized as follows: each dose contains 20-50 μgm ofHB_(s) Ag particle-asociated protein containing 10⁸ -10¹² Dane particles(preferably 10¹⁰ -10¹²); there are also additionally an equivalent orgreater number of filamentous particles. It should be understood thatthe vaccine contains only those large (30-50 nm) particles in which thee-antigen is free, i.e., unassociated with the corresponding anti-eantibody.

The vaccine is especially characterized, therefore, by the presence offree e-antigen on Dane or filamentous particles. However, the vaccinecan also contain e-antigen in a soluble form in the physiologicallycompatible medium which is employed.

The presence of the e-antigen can be detected by a number of methods;the following is adequate for the purposes of this invention:

INSENSITIVE TEST TO DETERMINE PRESENCE OF FREE E-ANTIGEN

The center well of an agar gel diffusion plate, preferably but notnecessarily made with 0.5-0.7% agarose in 0.1 M NaCl, 0.01 M Tris bufferpH 7.2, 0.001 M EDTA containing 2.0 w/v Dextran 250, 0.1% w/v protaminesulfate, is filled with anti-e antiserum having specificity similar tothat of the antiserum provided to us by Dr. Lars Magnius, and availablefrom the Blood Derivatives Division of The New York Blood Center.Alternative peripheral wells are filled with either reference e-antigen(provided by Dr. Magnius), or similar to it by agar gel diffusion"identity test", or test serum. A precipitin line appearing within 3days of incubation at room temperature, 30° or 37° C., which shows areaction of identity with the reference antigen, indicates presence oflarge quantities of e-antigen. The characteristics of HBsAg carriers asa function of e-antigen and/or antibody is set forth in Tables 1 and 2.

                  TABLE 1                                                         ______________________________________                                        CHARACTERISTICS OF HBsAg CARRIERS                                             AS A FUNCTION OF PRESENCE OF "e"-ANTIGEN                                      OR ANTI-"e" ANTIBODY                                                                         RESULTS OF TESTING                                                            BY AGAR GEL DIFFUSION                                          CHARACTERISTICS  "e"-Ag +    Anti-"e" +                                       ______________________________________                                        Proportion of Blood Donor                                                                      1 - 5%      30 - 70%                                         (asymptomatic HBsAg                                                           Carriers)                                                                     Proportion of HBsAg                                                                            20 - 70%     5 - 20%                                         carriers on chronic renal                                                     dialysis units                                                                Chronic Hepatitis found                                                                        Most        Very Few                                         by Liver Biopsy                                                               Chronic hepatitis                                                                              Most        Very Few                                         suggested by elevated                                                         SGPT levels                                                                   Infective to contacts                                                                          Very        Probably not                                     or offspring                 at all                                           ______________________________________                                    

                  TABLE 2                                                         ______________________________________                                        CHARACTERISTICS OF HBsAg CARRIER                                              PLASMA AS A FUNCTION OF PRESENCE                                              OF "e"- ANTIGEN OR ANTI-"e" ANTIBODY                                                         RESULTS OF TESTING                                                            BY AGAR GEL DIFFUSION                                          CHARACTERISTICS  e-Ag +      Anti-e +                                         ______________________________________                                        Dane particles in                                                                              10.sup.8 - 10.sup.12 /ml                                                                  <10.sup.6 *                                      Plasma                                                                        HBsAg associated Detectable  Absent                                           DNA polymerase in                                                             plasma                                                                        HBcAg detectable Detectable  Absent                                           in plasma by RIA or                                                           IAHA Tests                                                                    HBsAg Titer      10.sup.13 - 10.sup.14                                                                     10.sup.12 - 10.sup.13                                             particles/ml                                                                              particles/ml                                     ______________________________________                                         * None seen by electron microscopy                                       

METHOD OF PREPARING VACCINE

The method by which the vaccine is obtained is generally along the linesof patent application Ser. No. 426,825, U.S. Pat. No. 3,951,937 theentire disclosure of which is hereby specifically incorporated herein.The critical phase is the first step, namely the use starting materialcomposed of plasma obtained from chronic HB_(s) Ag carrier who havee-antigen(s) detectable by the above insensitive test. Such plasma ofcourse also contains abundant Dane particles and filaments free ofcombined anti-e antibody (see above). Such plasma may be suitablyobtained from clinically-well human chronic HB carriers, oralternatively from chimpanzees which have been naturally or artificiallyinfected with HB virus, and have become chronic e-antigen positiveHB_(s) Ag carriers. Generally speaking, plasma will be employed whichcontains HBsAg of the desired sub-type or sub-types. The starting plasmamust contain e-antigen detectable by the insensitive methods set forthabove and must contain no detectable anti-e antibody detectable by theabove method. Such plasmas are relatively rare but are essential to theprocess.

Given starting material of the desired characteristics, as describedabove, a simple method for the isolation of the e-antigen-containinglarger particle fractions, together with some contaminating smallerHB_(s) Ag-containing particles, and with only minor contamination ofserum proteins is provided by the following process:

1. Differential precipitation of antigen with polyethylene glycol; and

2. Absorption of contaminants by hydroxylapatite under conditions whichprevent adsorption of the larger HB_(s) Ag-containing particles, e.g. 24to 50 nm.

The resulting product can be readily concentrated, stabilized with smallquantities of human serum albumin, suspended in a physiologic vehicleand rendered non-infectious by combination of viral inactivationtechniques well known to the art of vaccine production. Immunogenicquantities are then injected at intervals sufficient to provide anadequate and long-lasting anti-e as well as anti-HB_(s) response. Suchimmunization is expected to provide protection against both high and lowdose infections with hepatitis B virus. Protection against such low andhigh dose infections by the known hepatitis B vaccine is difficult, ifnot impossible.

DETAILED DESCRIPTION OF INVENTION Source Material

Plasma used as source material for the purification procedures detailedbelow is obtained by conventional plasmaphoresis procedures from chronicHB_(s) Ag carriers. These may be humans or animal species such aschimpanzees in which the chronic HB carrier state can be induced. Thechimpanzee offers the practical advantage that it can be infected withhuman hepatitis B strains of any desired immunologic sub-type, and candevelop chronic carrier state infections which in our experience showparticularly high titers of HB_(s) Ag and are frequently associated withhigh concentrations of both Dane particles and e-antigen. Furthermore,these animals can be conveniently plasmaphoresed at frequent intervalswithout damage to their health or reduction in HB_(s) Ag or e Ag contentof their plasma.

The plasma pool selected for use as a starting material for this vaccinemust have:

A. e Ag detectable by an insensitive method such as agar gel diffusion.It should be noted that only 1-5% of chronic HB_(s) Ag carriers foundamong blood donor populations satisfy this criterion.

B. no detectable anti-e antibody. It should be noted that the majorityof HB_(s) Ag carriers found among blood donor populations have anti-eantibody. If such plasma were pooled with the desired plasma it wouldresult in precipitation of the e-antigen-containing particles, and wouldprevent their isolation by the methodology described.

C. dane particles readily detectable by conventional techniques ofelectron microscopy (negative staining) preferably in largeconcentrations.

D. sub-type antigenic composition of HB_(s) Ag which includes the majorsub-types encountered in the geographic region for which vaccine isdesigned. For example, a vaccine to be used in southeast Asia shouldpreferably contain a predominance of the r sub-type, whereas a vaccinedesignated for use in Europe or the United States should preferablycontain HB_(s) Ag predominantly of the w sub-type.

E. plasma from a sufficient number of donors, representing a mixture ofthe sub-types prevalent in the geographic area for which the vaccine isdesignated, to provide broad immunologic coverage. In most cases aminimum of 10 donors would be desirable. The use of "hybrid strains"such as HB_(s) Ag/adywr offers obvious advantages. Such hybrid strainscan be propagated in the chimpanzee.

F. the starting material should be frozen at -70° C. within 4 hours ofcollection and should be held at 4° C. during the interval prior tofreezing.

G. starting material should be bacteriologically sterile and should befree of adventitious viruses detectable by conventional techniques ofvirus isolation (inoculation of embryonated eggs, tissue cultures,suckling mice, guinea pigs and rabbits), as currently practiced in thequality control of other viral vaccines.

As indicated above, the purification can be conducted in the manner ofSer. No. 426,825, mentioned, supra. In such instance the fluid bloodmaterial-containing antigen is purified to remove impurities from theantigen-containing material by initially maintaining the pH of the bloodmaterial within the approximate range of 4.4 to 4.7. This results in asmall amount of precipitate which, together with remaining cells andcell debris can be removed by moderate speed centrifugation, e.g., 20minutes at 10,000 rpm. Thereafter, the material is admixed with 2.5 to4.5 weight percent of polyethylene glycol, preferably 3.0 to 4.5. Thelower concentrations, say 3-4%, can selectively precipitate largeparticle forms and therefore, are advantageous under certaincircumstances. The pH is maintained at 4.4 to 4.7. The weight percent ofthe polyethylene glycol is based upon the total weight of the mixture.This preciptates hepatitis B-containing antigen material of the largeparticle size, e.g., 30 to 50 nm, as well as a proportion of the knownshell-like hepatitis B surface antigen-containing material. Thepecipitate is recovered, and an amount of water is added such as topresent an intermediate fluid material having an antigen concentrationthe same or higher as in the original blood material. The pH of theintermediate fluid material is adjusted in the range of about 4.9 to5.1, optimally 5.0, whereby there is formed a precipitate containingproteinaceous material and polyethylene glycol and a fluid phasecontaining type B hepatitis antigen further characterized by thepresence of Dane particles and/or filamentous material. The fluid phaseis separately recovered, and the pH is adjusted to be in the range ofapproximately 4.4 to 4.7. The fluid phase is thereafter admixed, whileit is maintained in this pH range, with 3.0 to 4.5 weight polyethyleneglycol, based on the total weight of the mixture, to produce aprecipitate containing:

A. purified hepatitis B surface antigen containing particles ofshell-like configuration of particle size between 20 and 30 nm; and

b. (1) Filamentous particles containing HB_(s) Ag and e-antigen which isfree of any anti-e antibody, said filamentous particles being of adiameter between 20 and 50 nm, and

(2) Dane particles of generally spherical configuration, being of aparticle size of 40 to 50 nm, and having a lipoprotein outer shellcontaining HB_(s) Ag and e-antigens and containing a core, containingDNA, DNA polymerases and HBcAg.

The purified antigenic material is recovered. Thereafter, it can betreated to inactivate the virus, whereby the material is in a form fordilution with a physiologically acceptable medium.

Preferably, in such a separation the temperature of each of the mixturesafter each of the mixing steps is maintained in the range of 0° to 8° C.during the production of the precipitates. The purification step isfacilitated if recovery is augmented by the use of centrifugationfollowing the precipitation steps. Polyethylene glycol which is employedcan be used as such, or in the form of an aqueous solution, preferablyone which is about 30% by weight polyethylene glycol. Generally, thispolyethylene glycol is one which has a molecular weight in the range of500 to 50,000, preferably about 6,000.

Further purification can be effected by subjecting the purified antigensto adsorption on hydroxylapatite utilizing batch or columnchromatographic procedures. In the latter instance, the partiallypurified antigens are passed through a chromatographic column containinghydroxylapatite to which they adsorb. Further purified antigens are thenrecovered using a multi-, e.g., about three-step, elution procedure.Particles of particle size 25 to 50 nm are recovered separately from afraction of particles of smaller size (16-22 nm) and from the bulk ofserum proteins.

INACTIVATION OF INFECTIVITY

Following purification, the large particle fraction can be anticipatedto be infectious. It is, therefore, necessary that chemical and physicalprocedures be employed to effectively inactivate any and allinfectivity. Hepatitis virus is known to be very stable. This probablyresults in part from the small size of its nucleic acid, as well asother properties of its tertiary and quaternary structure andmorphologic organization.

Inactivation can be carried out by any combination of the variousconventional techniques known to the art of vaccine manufacture. The useof combinations is expected to eliminate "resistant fractions" which maybe expected with any single inactivation technique. These are destroyedby a second or third technique having a different principle ofinactivation from that formerly applied. This is the conventionalapproach for avoiding resistant fractions, e.g., in the treatment ofcertain bacterial infections, where drug resistant variants are common,by the use of combination antibiotic therapy. A schematic representationof this principle is illustrated in FIG. 3.

Any combination of presently well known techniques of inactivation whichsatisfy this criterion can be employed. The following, preferably incombination, is suggested:

1. Irradiation from a cobalt source with 2,500,000 rads;

2. Treatment with formalin at a concentration of 1:2,000 for 4 days at37° C. using conventional techniques. The vaccine is clarified byfiltration through a Millipore 0.22 μ filter, or equivalent, to removeany aggregates prior to the addition of formalin or β-propriolactone;

3. Use of β-propriolactone under conditions currently used forinactivation of rabies vaccine.

After inactivation the final product is diafiltered with an Amicon PM30filter, or equivalent, against 0.9% NaCl, containing 1:10,000Thiomerosal, and any desired conventional stabilizer, to remove any lowmolecular weight compounds used for or resulting from the inactivationprocedures, and is then sterile filtered and asceptically dispensed intosterile vials for lyophilization, or freezing, as preferred. Thiomerosalis sodium ethyl mercurithiosalicylate, a conventional antibacterialpreservative. The Amicon PM30 filter is a cellulose filter having anaverage pore diameter which nominally excludes molecules having amolecular weight of greater than 30,000.

SAFETY TESTING

Thereafter, the batch is tested to determine that it is safe, i.e., freeof infectivity. Until such time as there is a tissue culture techniqueor other standard test to determine the safety of HB and related virusinfectivity, it is necessary that each batch of vaccine made for humanuse be tested by inoculation of HB seronegative susceptible animals,such as chimpanzees or gibbons. At least four seronegative animals (twoinoculated with 5×20 μgm doses and two inoculated with 500×20 μgm, i.v.)should be used for this purpose. A satisfactory batch will not result ineither hepatitis, HB_(s) Ag production, or an anti-HBc response, eachdetected by the most sensitive methods currently available, in anyinoculated animal. The safety, i.e., lack of infectivity, can readily bedetermined by such a technique. Of course larger numbers of animals willbe used for evaluation of the early batches prior to clinical trials.

POTENCY TESTING

Vaccine is adjusted to a concentration of generally about 5 to 100 μgm,preferably 20 to 50 μgm of HB_(s) Ag-associated protein per dose, togive suitable immogenicity. Generally speaking, the quantity ofe-antigen-containing particles in such dose will amount to about 2 to 5μgm. Potency is controlled by determining the minimal concentration ofeach batch of vaccine which gives a definite specific anti-HB_(s) andanti-e response in guinea pigs, mice, or other suitable test animals.

Finally, the vaccine must have sufficient potency to provide ananti-HB_(s) titer of at least 1:100 by passive hemagglutination(standardized by tests on a frozen anti-serum control) in at least fourchimpanzees immunized with two doses of the standard vaccine inaccordance with the recommended schedule; and furthermore, anti-HB_(s)must remain detectable at a titer of greater than 1:10 for at least 1year following the onset of immunization of these chimpanzees. Inaddition, the chimpanzees must respond with a measurable anti-eresponse.

DOSAGE ADMINISTRATION AND UTILIZATION

Vaccine can be administered by sub-cutaneous or intramuscular injection.While the preferred route has not been yet determined, it is believedthat intramuscular injection is preferred. The frequency ofadministration is usually about two doses 1 month apart, followed by abooster at 6 months to 1 year after primary immunization. Of course, thedosage will depend upon the size of the host animal being inoculated.The subsequent doses or the booster will depend upon the level ofantibody in the blood as a result of the initial immunization.Licensable adjuvants conventionally employed in vaccine manufacture canbe utilized.

The vaccine is recommended for all persons at risk of developinghepatitis B infection, and particularly those at especially high risk,such as patients and staff on hemodialysis units, medical personnel,persons of tropical populations and those visiting the tropics. In thecase of tropical populations, particularly in Africa, Asia, themediterranean region and South America, where a high incidence ofhepatitis B infections has been consistently observed, the vaccineshould be administered sufficiently early in life to prevent acquisitionof chronic carrier state infections which tend to occur in these regionsduring the first 5 years of life.

When adequate supplies are available, and effectiveness has beendocumented, the vaccine will be useful for all persons not alreadyprotected against hepatitis B infections as a result of prior immunity.

The particular importance of hepatitis B vaccine lies in its role inprevention of the chronic hepatitis B carrier state with its attendantrisk of development of chronic liver diseases, such as chronic activehepatitis, cirrhosis and hepatoma. The association of a chronichepatitis B carrier state and these diseases is now abundantlydocumented, and the role of chronic hepatitis B as a cofactor in theetiology of these conditions is also established. [Prince, A.M., in TheLiver: Normal and Abnormal Functions, edited by F. F. Becker (M. Dekker,New York) 1975].

The use of the new hepatitis B-containing vaccine in the long run canhave a significant effect in the prevention of these chronic liverdiseases which are so extraordinarily prevalent in those regions,primarily in the tropics, where chronic hepatitis B carrier state isunusually frequent. For example, in Senegal where 14% of the populationchronically carry HB_(s) Ag, the attack rate of primary liver cancer is50 to 100 times as high as that observed in the United States, whereonly an average of approximately 0.3% of the population are chroniccarriers of hepatitis B virus.

It should be emphasized that the approaches to purification of the largeparticle fractions described in the present invention are not the onlyapproaches available for this purpose. Variations designed to meetindividual needs and preferences can be employed, and these includethose known or to be hereafter developed. What is important and what iscontemplated by the scope of the invention is the use of large particle,e-antigen-containing fractions which have routinely been discarded byprior art researchers. Thus, all vaccine containing such particles; orspecific soluble components of such particles; or syntheticantigenically specific sub-units thereof, are contemplated as these areuseful in an active immunization program against e-antigen or similarDane particle specific antigens.

BRIEF DESCRIPTION OF DRAWINGS

Referring to the drawings herein,

FIG. 1 shows in a graphic fashion the diverse morphologic formsassociated with hepatitis B surface antigen, it being understood thattogether with the hepatitis B surface antigen in accordance with thepresent invention there is e-antigen which can be present on Daneparticles which contain hepatitis B core antigen (HBcAg), these Daneparticles being characterized by deoxyribonucleic (DNA) and itspolymerase. The figure shows the possible antibodies includingantibodies of hepatitis B surface antigen, antibodies of hepatitis Bcore antigen and antibodies of e-antigen;

FIG. 2 graphically shows the differential effects of antibodies ofe-antigen and antibodies of hepatitis B surface antigen in theneutralization of Dane particles as a function of exposure dose. Fromthis it is seen that the antibodies generated from a HBsAg vaccine freeof e-antigen are insufficient to effect total neutralization of theantigen containing sites. However, where a high dosage of antigen isinvolved, the antibodies generated from a vaccine characterized by theaddition of e-antigen are sufficient to neutralize virtually all of theantigen sites, thereby indicating that the presence of e-antigen in avaccine materially affects the extent to which neutralization of allantigens takes place;

FIG. 3 graphically illustrates the fact that with any given inactivationprocedure a minor proportion of the infectious virus will be resistantto inactivation. However, when different inactivation techniques areemployed sequentially, these resistant fractions can be inactivated;

FIG. 4 is a figure reflecting the chromatographic results obtained bythe column chromatographic fractionation of a PEG precipitated HBsAgmaterial on a 500 ml column of hydroxylapatite. The data was derived byresuspending PEG precipitated HBsAg/e in 50 ml volume of liquid and itwas chromatographed on a 500 ml column of hydroxylapatite. The antigenwas eluted with a discontinuous gradient of 1 liter each of 0.02 M, 0.05M and 0.10 M sodium phosphate buffer pH 7.2. Fractions of 10 ml volumewere collected and alalyzed for protein by O.D. at 280 nm (•--•--•) andfor HBsAg by CEP (x--x--x );

FIG. 5 depicts the results of cellulose acetate electrophoresis ofpooled concentrated samples of HBsAg/e which has been purified bychromatography on hydroxylapatite (Table 4). The strip was scanned in aBeckman microzone densitometer after Ponceau red staining.

Line A: the original HBsAg/e containing plasma.

Line B: the resuspended PEG precipitate of HBsAg/e.

Line C: column eluate of HBsAg/e -- Pool III.

Line D: column eluate of HBsAg/e -- Pool II.

Line E: column eluate of HBsAg/e -- Pool I;

FIG. 6 is a graphic drawing showing the results of the purification of aPEG precipitated HBsAg/e material by column chromatography onhydroxylapatite. A 200 ml volume of resuspended PEG precipitate ofHBsAg/e was applied on a 1000 ml volume column of hydroxylapatite. Theantigen was eluted with a discontinuous gradient of 0.5 liters of 0.02 Mand 1 liter of 0.05 M sodium phosphate buffer pH 7.2. Fractions of 12 to13 ml were collected and analyzed for proteins by O.D.;

FIG. 7 is a graphic drawing showing the results of column chromatographyperformed on a HBsAg/e material on hydroxylapatite. Pool I of HBsAg/eeluted from hydroxylapatite II (FIG. 6) was concentrated to 75 ml andreapplied on a 600 ml column of hydroxylapatite. The antigen was elutedwith a discontinuous gradient of 1 liter each of 0.02 M, 0.05 M and 0.10M sodium phosphate buffer pH 7.2. Fractions of 18 ml volume werecollected and analyzed for proteins by O.D. at 280 nm (•--•--•), forHBsAg by CEP (x--x--x) and RIA (o--o--o);

FIG. 8 shows the results of immunoelectrophoresis of fractions ofHBsAg/e which has been eluted from hydroxylapatite, theimmunoelectrophoresis being conducted in agrose gel. The samples weretested with antiserum prepared by immunization of rabbits with normalchimpanzee plasma.

Samples: (1) Pool I; (2) Pool II; (3) Pool III; (4) Pool IV; (5) Pool V;(6) Pool VI; (7) Pool VII-VIII;

FIG. 9 shows the results of polyacrylamide gel electrophoresis ofpurified preparations of HBsAg/e subjected to Coomassie blue staining.The samples were run in a 7.5% SDS-acrylamide gel under the conditionsdescribed above. The gels after Coomassie blue staining were scanned ina Joyce-Loebl microdensitometer:

A: 20-22 nm spherical particles of HBsAg

B: 22-28 nm spherical particles of HBsAg

C: hbsAg/e particles composed mostly of filaments and Dane particles;

FIG. 10 shows the result of polyacrylamide gel electrophoresis ofsamples of purified HBsAg/e run parallel with those in FIG. 9. The gelsafter Schiff base staining were scanned in a Joyce-Loeblmicrodensitomer;

FIG. 11 is a series of electron microscopes of HBsAg/e particles whichhave been purified by batch-wise elution from hydroxylapatite and ratezonal centrifugation. Pictures were taken after negative staining withphosphotungstate in a JEOL 100B cleatron microscope at 67,000magnification.

a: Rate zonal centrifugation -- Pool I: Dane particles and largefilaments.

b: Rate zonal centrifugation -- Pool II: filaments 22-28 nm sphericalparticles.

c: Rate zonal centrifugation -- Pool III: 20-28 nm spherical particles;

FIGS. 12A and 12B are graphic representations of data revealed bythin-layer isoelectric focusing of pooled concentrated samples ofHBsAg/e in Sephadex G-75 suspended in 1% pH 3-6 ampholine. The fractionswere analyzed for HBsAg by RIA (•--•--•) and for pH (x--x--x ) asdescribed above.

A: pool I: 22-28 nm spherical particles of HbsAg.

B: pool II: 22-28 nm spherical particles, filaments and a few Daneparticles; and

FIGS. 13 show additional data obtained from thin-layer isoelectricfocusing of the same material with 1% pH 3.5-10.0 ampholine of pooledconcentrated samples of HBsAg/e (Table 4) after Triton X-100 treatment.The fractions were analyzed for HBsAg by RIA (•--•--•) and for pH(x--x--x ) as described above.

A: pool I: 22-28 nm spherical paeticles.

B: pool II: 22-28 nm spherical particles, filaments and a few Daneparticles.

EXAMPLES

In order to more fully illustrate the nature of the invention and themanner of practicing the same, the following examples are presented.

Above there are outlined the general steps for purification of hepatitisB surface antigen from human or chimpanzee plasma by polyethylene glycolprecipitation (PEG) followed by purification on hydroxylapatite. Setforth below are the results of the application of these procedures forlarge scale purification and fractionation of chimpanzee hepatitis Bsurface antigen containing particles having e-antigen. The samples atindividual steps of purification were analyzed in detail usingelectrophoretic and chromatographic techniques. Comparative studies wereperformed with fractions of different morphological forms of purifiedchimpanzee HBsAg associated particles by thin-layer isoelectric focusingand polyacrylamide gel electrophoresis.

EXAMPLE 1: PURIFICATION AND CHARACTERIZATION OF HB ASSOCIATED PARTICLESFROM 7.8 LITERS OF PLASMA FROM A SINGLE HBsAg CARRIER CHIMPANZEE.Materials and Methods

Source of HBsAg:

The antigen was purified from pooled plasma of a carrier chimpanzeepreviously inoculated with human HBsAg-positive plasma of the adwsubtype. The e-antigen was present on the surface of identifiable Daneparticles in the plasma used.

Purification of HBsAg and e-Antigen:

HBsAg and e-antigen was isolated from plasma by polyethylene glycol(PEG) precipitation. The resuspended precipitate of HBsAg and e-antigenwas purified by column chromatography on hydroxylapatite. The finalsteps of purification involved density gradient centrifugation. Themethods of purification were essentially the same as outlined in Ser.No. 426,825, hereby incorporated by reference. The procedures arebriefly described as follows:

Precipitation of HBsAg and e-Antigen by PEG:

In a pre-purification step, 7,800 ml of HBsAg and free e-containingplasma was adjusted to pH 4.6 and a 30% solution of PEG in distilledwater was added to approximately 2% concentration. The solution wasstored overnight at 4° C. and clarified by centrifugation. The HBsAg inthe supernatant was precipitated by raising the PEG concentration to 4%.The supernatant after overnight storage of the sample at 4° C. wasdecanted and discarded. The sediment of HBsAg (containing e-antigen) wasresuspended to 500 ml in distilled water. The bulk of PEG with someaccompanying contaminants was precipitated by adjusting the solution topH 5.0 and the precipitate was removed by centrifugation. The clearsupernatant was readjusted to pH 4.6, and the HBsAg and particleassociated e-Ag (HBsAg/e) was precipitated by adding a 30% solution ofPEG to a final concentration of 4%. The HBsAg/e was removed bycentrifugation after overnight storage of the sample at 4° C. Finally,the precipitate of HBsAg/e was resuspended to 320 ml in distilled water.

Column chromatography on Hydroxylapatite:

In a trial experiment, 50 ml of resuspended PEG precipitate of HBsAg/ewas applied to a 6.5 × 16.5 cm. column of hydroxylapatite and elutedwith a discontinuous gradient of phosphate buffer. In a subsequentexperiment 200 ml of the sample was partially purified on a 6.5 × 35 cmcolumn of hydroxylapatite. The HBsAg eluted with the first protein peakwas passed through a hydroxylapatite column for a second time.

Density Gradient Centrifugation:

Fractions of HBsAg isolated by column chromatography were purified byrate zonal centrifugation. A discontinuous gradient was formed using 3ml of 60% and 7 ml of 40% sucrose in 0.02 M sodium phosphate buffer pH7.2 (containing 0.02% NaN₃). A 20 ml volume of sample was applied toeach tube and centrifuged in a Spinco SW 25.1 rotor at 18,000 rpm for 18hours. Fractions of 1 ml volume were collected by dripping from thebottom of tubes and analyzed for HBsAg. The peak fractions of antigenicactivity were pooled, thoroughly dialyzed and concentrated byultrafiltration using a membrane with a 30,000 MW cutoff (Amicon PM-30).The concentrated fractions of HBsAg were submitted to final steps ofpurification by isopycnic banding in a linear CsCl gradient and ratezonal centrifugation in a continuous sucrose gradient under conditionsnormally employed.

Detection Methods:

HBsAg was monitored by counter electrophoresis (CEP) or solid phaseradioimmunoassay (Ausria I or II, Abbott Laboratories). The proteinconcentration was estimated by measurements of O.D. at 280 nm and bymicro Kjehldahl technique. The methods used were the same as previouslydescribed in the above-identified patent application.

Criteria of HBsAg Purity:

The samples after each step of purification were analyzed by celluloseacetate electrophoresis, immunoelectrophoresis, and agar gel diffusiontests against polyvalent anti-normal human plasma protein antiserum.

Polyacrylamide Gel Electrophoresis:

Aliquots of samples containing 0.01 sodium phosphate buffer pH 7.2, 1%sodium dodecylsulfate, 1% β-mercaptoethanol and 10% glycerol were heatedfor 2 minutes in a boiling water bath, applied to 7.5% or 10% sodiumdodecylsulfate-acrylamide gels, and elto Maizel. The gel was stained forproteins by Coomassie Blue staining and for carbohydrates by Schiff basestaining according to the method of Glossman and Neville.

RESULTS

The results of purification of HBsAg/e positive sera by polyethyleneglycol precipitations are presented in Table 3. Apparently about 87% ofthe original plasma protein was removed in the first step ofprecipitation of HBsAg (which included pre-purification at 2% PEGconcentration). The final preparation after two successive PEGprecipitation steps showed quantitative recovery of HBsAg with only 4%of the original proteins.

                                      TABLE 3                                     __________________________________________________________________________    PURIFICATION OF POOLED CHIMPANZEE HBsAg BY                                    PEG 6000 PRECIPITATION                                                                                     TOTAL                                                           VOLUME                                                                              HBsAg   PROTEIN                                                                             % YIELD .sup.2                                                                       FOLD .sup.3                         SAMPLE         (ml)  CEP TITER .sup.1                                                                      (g)   HBsAg  PURIFICATION                        __________________________________________________________________________    Original HBsAg-Plasma                                                                        7,800 512     429.0 100    --                                  First 4% PEG Precipitate                                                                     500   7,080   55.0  89     6.9                                 Second 4% PEG Precipitate                                                                    320   10,000  16.6  80     20.7                                __________________________________________________________________________     .sup.1 Kjehldahl                                                              ##STR1##                                                                      ##STR2##                                                                 

Hydroxylapatite Chromatography I

In a preliminary experiment, a 50 ml volume of resuspended PEGprecipitate of HBsAg was fractionated by column chromatography onhydroxylapatite. The results of chromatography are illustrated in FIG.4. The eluate was pooled into seven fractions, which were dialyzedagainst 0.02 M sodium phosphate buffer of pH 7.2 (containing 0.02%sodium azide) and concentrated by ultrafiltration. The concentratedsamples were analyzed for proteins by O.D. measurements at 280 nm andfor HBsAg by CEP. The results of fractionation are summarized in Table4.

                                      TABLE 4                                     __________________________________________________________________________    HYDROXYLAPATITE CHROMATOGRAPHY I                                              PURIFICATION OF THE SECOND 4% PEG PRECIPITATE OF HBsAg                                              VOLUME                                                                              HBsAg   TOTAL .sup.1                                                                            % YIELD .sup.2                                                                        FOLD .sup.3             NO. SAMPLE            (ml)  CEP TITER                                                                             PROTEIN (mg)                                                                            HBsAg   PURIFICATION            __________________________________________________________________________    1   Second 4% PEG precipitate HBsAg                                                                 50    10,000  2,600.0   100     0                       2   Ottapatite I:                                                                           Pool I  4     10,000  63.2      B       3.3                     3             Pool II 6     10,000  87.2      12      3.6                     4             Pool III                                                                              5     10,000  183.9     10      1.4                     5             Pool IV 6     800     110.0     1       0.2                     6             Pool V  6     800     72.6      1       0.3                     7             Pool VI 10    20,000  984.0     40      1.1                     8             Pool VII                                                                              7     10,000  338.3     14      1.1                     __________________________________________________________________________     .sup.1 # 1 Kjehldahl                                                          #s 2-8 O.D. 280 nm E.sub.1cm.sup.0.1% = 1.143                                 ##STR3##                                                                      ##STR4##                                                                 

Cellulose acetate electrophoresis (FIG. 5) showed in comparison to theoriginal plasma (FIG. 5A) two protein bands in Fraction I (FIG. 5E) andFraction II (FIG. 5D) migrating between alpha₁, alpha₂ and betweenfibrinogen and beta globulin regions. Fraction III (FIG. 5C) showed twoadditional components in the beta globulin and albumin regions. Thesuspended PEG precipitate of HBsAg (at 24 × fold concentration) showedstrong lines between alpha₁ and alpha₂ region and in the origin(fibrinogen) and two additional bands in the albumin and gamma globulinregions (FIG. 5B).

As indicated in FIG. 4, HBsAg was separated into three peaks ofantigenic acitivity. The samples analyzed by electron microscopy showedmostly spherical particles of 22 to 28 nm in diameter in the first peak(Fraction I); variable size particles, including Dane particles andlarge filaments, in the second peak (fraction II). The third peak ofantigenic activity (Fraction VI) contained mostly 16 to 22 nm sphericalparticles.

Hydroxylapatite Chromatography II

A further 200 ml volume of the resuspended PEG precipitate of HBsAg wasfractionated on a 1000 ml volume column of hydroxylapatite. The resultsof chromatography are illustrated in FIG. 6. The fractions of HBsAg fromthe first protein peak (pool I: fractions 38 to 83) and the residualeluate (pool II: fractions 84-110) were each pooled and concentrated to75 ml by ultrafiltration.

Hydroxylapatite Chromatography III

The concentrated sample from pool I of the above separation wasrechromatographed on a 1000 ml volume column of hydroxylapatite. Theresults of this chromatography are illustrated in FIG. 7. The eluate waspooled into eight fractions according to the figure. The pooled sampleswere concentrated by ultrafiltration and washed with 0.02M sodiumphosphate buffer pH 7.2 (containing 0.02% sodium azide as describedabove.) The samples were analyzed for proteins and HBsAg and the resultsare summarized in Table 5.

                                      TABLE 5                                     __________________________________________________________________________    HYDROXYLAPATITE CHROMATOGRAPHY III                                            PURIFICATION OF POOL FROM HYDROXYLAPATITE CHROMATOGRAPHY II                                                    TOTAL .sup.1                                                    VOLUME                                                                              HBsAg   PROTEIN                                                                             % YIELD .sup.2                                                                        FOLD .sup.3                    SAMPLE             (ml)  CEP Titer                                                                             (mg)  HBsAg   PURIFICATION                   __________________________________________________________________________    O Hydroxylapatite II:                                                                      Pool I                                                                              75    10,000  706.0 100     --                             O Hydroxylapatite III:                                                                     Pool I                                                                              5     8,000   51.8  5.3     0.12                                        Pool II                                                                             5     2,000   14.2  1.3     0.65                                        Pool III                                                                            4     1,600   11.4  0.9     0.56                                        Pool IV                                                                             4     3,200   7.3   1.7     1.64                                        Pool V                                                                              12    16,000  181.6 25.6    1.00                                        Pool VI                                                                             7     8,000   79.6  7.5     0.61                                        Pool VII                                                                            45    4,000   332.8 24.0    0.51                           __________________________________________________________________________     .sup.1 O.D..sub.280 nm E.sub.1cm.sup.0.1% = 3.16                              ##STR5##                                                                      ##STR6##                                                                 

Analyses of samples for contaminants by immunoelectrophoresis (FIG. 8)showed a very weak precipitin line in fraction I, a stronger line infractions II to VI and a heavy broad line in the pooled fractionVII-VIII all migrating between the alpha₂ and albumin regions.

Electron microscopy of samples showed mostly spherical particles of 22to 28 nm in diameter in fractions I to III, spherical particles andfilaments in fractions IV to VI, mostly small spherical particles and afew filaments in fraction VII and VIII.

The concentrated samples from the previous step were submitted tocomplete purification by density gradient centrifugation under theconditions described under Methods.

Purified preparations of HBsAg composed mostly of 20-22 nm sphericalparticles, 22-28 nm spherical particles, variable size filaments andDane particles, each containing 100 μgm of protein were analyzed forpolypeptide composition by polyacrylamide gel electrophoresis (FIG. 9).Apparent quantitative differences can be observed in protein peaks ofindividual samples. Further differences are found in the peak 5 whichshows only as a small shoulder in the preparation of 20-22 nm particles(FIG. 9A) but it is distinct in the preparation of 22-28 nm particles(FIG. 9B) and in the sample containing mostly filaments and Daneparticles (FIG. 9C). Aliquots of 20-22 nm (FIG. 10A) and 22-28 nmspherical particles (FIG. 10B) run in parallel to the above samples weretested for carbohydrates by Schiff base staining. It is evident thatpeaks P₁, P₂, P₄ and P₆ contain carbohydrates.

The low molecular weight carbohydrate peaks (on the right of FIG. 10)probably represent glycolipids since they do not show by Coomassie bluestaining. The approximate molecular weights of the polypeptides arepresented in Table 6.

                                      TABLE 6                                     __________________________________________________________________________    MOLECULAR WEIGHT (X 10.sup.3) OF POLYPEPTIDES                                 OF HBsAg/e PARTICLES                                                          SUBTYPE: adwx                                                                 __________________________________________________________________________    Peak Number                                                                              P1  P2  P3  P4  P5  P6  P7   P8   P9                               Chimpanzee HBsAg/e                                                                       23-24                                                                             26-27                                                                             --  44-45                                                                             55  68-69                                                                             103-105                                                                            108-110                                                                            115-118                          __________________________________________________________________________

A vaccine is prepared from the large particle fractions obtained aboveby addition of human serum albumin at 0.5 mg/ml followed by inactivationserially by cobalt irradiation (2.5 million rads) formalin inactivationusing conventional procedures (37° C., 96 hours 1:2,000 concentration)followed by use of β-propiolactone using conventional procedures as inrabies vaccine manufacture.

EXAMPLE 2: A MODIFIED IMPROVED PEG PROCEDURE FOR ISOLATION OF HBsAg/e

A modified procedure for PEG precipitation was applied to 2.6 liters ofpooled HBsAg containing plasma from a single carrier chimpanzeecontaining e-antigen in an uncombined, unprecipitated form. The plasmawas adjusted approximately to pH 4.6 and immediately a 30% solution ofPEG was added to a final concentration of 2%. The solution was chilledon ice for 30 minutes (instead of overnight at 4° C. as in the previousexample), and the precipitate was removed by centrifugation.

The supernatant, containing the HBsAg, was brought to a PEGconcentration of 4% (instead of 4.5% as in the previous example) byaddition of 6.6 parts per 100 of a 30% solution of PEG at roomtemperature. The suspension was again chilled on ice for about 30minutes and the precipitated HBsAg/e was removed by centrifugation. Theprecipitate was resuspended to 200 ml in distilled water. The bulk ofPEG and some accompanying contaminants were removed by adjusting the pHto 5.0, chilling the solution on ice for 30 minutes, and removing theresultant precipitate by centrifugation. The pH of the clear supernatantfrom the previous step was readjusted to 4.6 and 30% PEG was added to afinal concentration of 3% (instead of 4.0-4.5% as previously employed).The material was again chilled on ice for about 30 minutes and theprecipitate of HBsAg/e was removed by centrifugation.

The precipitate from the previous step, containing HBsAg/e, wassuspended in 100 ml distille water. The bulk of the PEG was precipitatedby again adjusting the pH to 5.0. After overnight storage at 4° C. thesuspension was clarified by centrifugation. To the clear supernatant,containing HBsAg/e, 0.5 M sodium phosphate pH 7.2 was added to a finalconcentration of 0.02 M and the final volume was adjusted to 120 ml withdistilled water.

The results of this purification, summarized in Table 7, show more than33 fold purification of HBsAg (against 24 fold purification in theprevious procedure)

                                      TABLE 7                                     __________________________________________________________________________    PURIFICATION OF HBsAg BY PEG 6000 PRECIPITATION                                                    HBsAg                                                                              TOTAL PERCENT                                                      VOLUME                                                                              1/CEP                                                                              PROTEIN                                                                             YIELD FOLD                                    SAMPLE         (ml)  TITER                                                                              (g) .sup.1                                                                          HBsAg .sup.2                                                                        PURIFICATION .sup.3                     __________________________________________________________________________    Original Plasma                                                                              2600   80  130.0 100.0 0.0                                     First 4% PEG Precipitate                                                                     1200   160 22.8  92.3  5.3                                     Second 3% PEG Precipitate                                                                     120  1600 3.6   92.3  33.3                                    __________________________________________________________________________     .sup.1 Kjehldahl                                                              ##STR7##                                                                      ##STR8##                                                                 

The factors which contribute to the improved procedure (example 2) are:

1. Chilling of solutions on ice for 30 minutes instead of overnightshortens the purification to 1 working day as compared to several daysin the method of example 1.

2. Shortening the time of cooling leads to more specific precipitationof HBsAg/e and less contamination with plasma proteins.

3. Reducing the volume of sample after the first precipitation ofHBsAg/e requires smaller volumes of PEG for selective precipitation insubsequent steps, and allows the second precipitation at pH 5.0 toremove the bulk of PEG from the sample.

Vaccine containing albumin is produced by the inactivation steps ofExample 1.

EXAMPLE 3: FRACTIONATION OF HGsAg/e BY BATCHWISE TREATMENT WITHHYDROXYLAPATITE.

The resuspended PEG precipitate of HBsAg/e was (example 2) treatedbatchwise with hydroxylapatite instead of by the column chromatographyprocedure. A 500 ml volume of packed hydroxylapatite (Bio-Rad Labs) wassuspended in 800 ml of 0.02 M sodium phosphate buffer pH 7.2, and, afteraddition of the above preparation of HBsAg/e, was stirred on a magneticstirrer for 30 min. at room temperature. The slurry was divided into twoequal aliquots and centrifuged in 1 liter buckets in the Sorvall RC-3centrifuge at 4,000 rpm for 10 min. The supernatant was decanted and thesediment was successively washed with 1 liter each of 0.02 M and 0.05 Msodium phosphate buffer pH 7.2. The eluates were pooled, concentrated byultrafiltration using a PM-30 membrane and diafiltered with 0.02phosphate buffer of pH 7.2, 0.02% NaN₃, and adjusted to 60 ml with thesame buffer.

The concentrated sample was further purified by rate zonalcentrifugation in a discontinuous sucrose gradient.

The separated HBsAg/e particles were pooled into three fractions,dialyzed and concentrated by ultrafiltration as described above. Thesamples studied by electron microscopy showed mostly large filaments andDane particles in Fraction I (FIG. 11a); filaments, Dane particles and22-28 nm spherical particles in Fraction II (FIG. 11b) and 20-28 nmspherical particles in Fraction III (FIG. 11c).

Batchwise treatment of the resuspended PEG precipitate of HBsAg/e hasthe advantage over column chromatographic procedures describedpreviously, in that fractionation is realized within a day as comparedto 5 days required for elution by colum chromatography. The vaccine isprepared by the procedures of Example 1 using albumin and the statedinactivation procedures.

EXAMPLE 4: DISAGGREGATION OF HBsAg/e PARTICLES WITH IONIC AND NONIONICDETERGENTS.

HBsAg/e particles were treated at room temperature (for 5 min to 2hours) with the following detergents: 0.5-2% Nonidet NP-40 (a detergent)(Shell Oil Co.), 0.5-2% Tween 80 (surfaceactive agent), 5-10% TritonX-100 (organic surfaceactive agent), 0.1-1.0% sodium dodecylsulfate,etc. The objectives were: (1) potentiation of antigenic acitivity indetergent treated samples, and (2) inactivation of infectivity of thesamples.

The optimal conditions of treatment were determined by analyses ofsamples before and after detergent treatment using thin layerisoelectric focusing. Individual components of disaggregated samples canbe isolated using various separation techniques. For example, molecularexclusion, adsorption and ion exchange chromatography ultracentriguationtechniques, various methods of electrophoresis, etc. One of the highlyefficient techniques is the method of zone convection isoelectricfocusing described below.

Isoelectric Focusing:

Thin layers were formed on Sephadex G-75 gell cellulose support, sold byPharmacia, Uppsala, Sweden in a 1% solution of pH 4.0-6.0 or pH 3.5-10.0ampholine according to the method of Radola. The plates were run in anLKB multipore apparatus. Prints were taken into Whatmann 3MMchromatography paper. Prints were divided along the line of separatedsamples and cut into 0.5 to 1 cm. zones. The individual zones werefurther cut into smaller pieces, moistened with 0.2 ml. of bufferedsaline and eluted with 0.2 ml. of normal (free of HBsAg and anti-HBs)human serum. A 200 ul aliquot from each eluate is used forradioimmunoassay. The strips between the separated samples are also cut,eluted with 0.4 ml of degassed distilled water and used for pHmeasurements. When two prints were taken, the first one was used forprotein staining and the second was cut for HBsAg radioimmunoassay andpH measurements. An aliquot from each sample is treated with 5 to 10% ofa non-ionic detergent (Triton X-100 ) for at least 20 minutes at roomtemperature before application to the gel and run under the abovedescribed conditions in comparison to a sample which has not beentreated with detergents.

Zone Convection Isoelectric Focusing:

An apparatus was constructed from lucite on the principles described byValment (U.S. Pat. No. 3,616,456). It consisted of 32 compartments of 50ml total volume. Certain modifications leading to improvements wereintroduced. The trough was filled with 50 ml. of solution containing 1%ampholine (LKB) of the desired pH range and 10% glycerol. The solutionin compartments No. 4, 5 and 6 (counting from the anode) was replacedwith 1.5, 3.0 or 4.5 ml. of the sample containing 1% ampholine and 10%glycerol. Electrofocusing was fun at 1,000-1,500 V for 4 days at 4° C.The fractions were separated from the individual compartments afterremoving the lid, clarified by centrifugation, and analyzed for proteinsby measuring O.D. at 280 nm., and for HBsAg by RIA and for pH.

Analysis of Triton X-100 Treated Preparations of HBsAg/e

Fractions of HBsAg/e particles isolated by column chromatography onhydroxylapatite (FIG. 4) were analyzed by thin-layer isoelectricfocusing under the conditions described above. A preparation composedmostly of 22-28 nm. spherical particles (FIG. 12A) and a heterogenousmixture consisting mostly of larger particles (filaments, Daneparticles) (FIG. 12B) showed a relatively low degree of heterogeneity inthe surface charge of the particles. A main peak of antigenic activitywas found at pH 4.8 and two minor peaks of activity were observed at pH4.0-4.1 and pH 4.4-4.6. The samples after Triton X-100 treatment showeda two-fold enhancement of antigenic activity determined by planimetry(FIG. 13). The enhancement of activity in other experiments varied from2-8 fold. Other than some residual antigenic activity observed in theoriginal low pH region (pH 1.4-5.0) the main peaks of antigenic activitywere found at a higher pH range of 5.3 and 5.8 to 5.9 and a minor peakat pH 7.1-7.2. E-antigen activity was found only in the pH region5.6-7.4. This antigen was found in this region by the insensitive agargel diffusion test. Thus the quantity of e-antigen in this region isclearly high. Indeed the radioimmunoassay peak at this region couldrepresent contamination of the ¹²⁵ I-"Anti-HBs" used in the Ausriatechnique (Abbott Laboratories) by a very small quantity of anti-e.

Thus disaggregation of HBsAg/e by non-ionic detergent Triton X-100 leadsto potentiation of antigenic activity. This has an obvious advantage forpreparation of antigenic material for active immunization. A furtheradvantage of detergent dissociation is the fact that this results inphysical dissociation of the HB particles.

Few if any particles survive intact after treatment with Triton X-100under the conditions described. This in itself is therefore an importantstep in the inactivation of infectivity. Furthermore, if desired, thisstep can be combined with procedures such as precipitation with anti-HBcantibody, and/or Deoxyribonuclease treatment, to remove all HBVassociated genetic material from the vaccine. This may prove desirablebecause of the (theoretical) possibility of oncogenicity.

This experiment thus reveals one method to purify e-antigen from HBsAg/eassociated particles by a combination of Triton X100 dissociation andzone convection isoelectric focusing. As equipment is now available(Roche Institute) for carrying out this technique on a multiliter scale,the approach described is suitable for preparation of an e- antigencontaining vaccine which is essentially free of conventional HBsAg. Sucha vaccine has obvious advantages as indicated above.

The final vaccine product is produced by the procedure of Example 1using human albumin and the stated inactivation procedures upon thedesired fraction after detergent association.

EXAMPLE 5: PURIFICATION OF SOLUBLE e-ANTIGEN FROM PLASMA OF HBsAg/eCHRONIC CARRIERS FOR USE AS A VACCINE

As indicated above (see FIG. 2) HBsAg/e positive plasma containse-antigen in a soluble (i.e. non particle associated, or MW <1,000,000)form. Indeed this form predominates in many such plasmas, and thereforecan provide an ideal source of e-antigen for immunization.

The properties of soluble e-antigen have been determined by Magnius,Ohori and ourselves in a preliminary manner as follows: density (CsCl)1.28-1.29; S₂₀,W = 11.6; Molecular weight = 150,000-900,000; Isoelectricpoint 5.5-6.6; elution from diethylaminoethyl cellulose at 0.15 M NaCl.

On the basis of these and other properties the following purificationmethod has been developed:

1. HBsAg/e plasma is first fractionally precipitated by PEG 6000 withremoval of HBsAg associated particles and some serum proteins byprecipitation with 5-9%, preferably 8% w/y, PEG 6000 at pH 7.0, 20° C.,followed by precipitation of e-antigen and some plasma proteins with PEGat 9-13% PEG concentration. Variations of the details of fractionalprecipitation, e.g., in pH, temperature, PEG concentration, as well asuse of the pH 5.0 precipitation step to remove PEG, can also be used.

2. The PEG precipitate is resuspended in distilled watter (1/10 volume)or suitable buffer and subjected to isoelectric focusing in 10%glycerol, containing 1-2% ampholytes of pH range 3.5-10.0.

3. The e-Ag peak is pooled, diafiltered on an Amicon PM 30 filter vs0.05 M phosphate buffer, 0.01 M Triton X 100 pH 7.6, and applied to aDEAE cellulose column equilibrated with the same buffer. Elution isperformed with a linear gradient of 0.0-0.5 M NaCl in the same buffer.E-Ag is eluted at about 0.15 M NaCl in a high state of purity.

4. After concentration by diafiltration as before, remaining impuritiesare removed, if desired by rate zonal centrifugation in 10-30% sucrosegradient, or equivalent density gradient, with recovery of the 11.6 speak.

5. Purified e-Ag is stabilized by addition of human serum albumin to aconcentration of Ca 0.5 mg/ml. The concentration and nature of colloidstabilizer is of course not critical.

Such preparations, at a concen tration of about 20 μgm per dose, may beused instead of the particulate of HBsAg/e vaccine, subject to the useof inactivation and sterilization steps as described herein, forpreparation of the HBsAg/e particulate vaccine.

Application Ser. No. 426,825 referred to hereinabove is assigned to theassignee of this present application.

What is claimed is:
 1. A method of manufacturing a vaccine againsthepatitis which comprises:A. obtaining the blood serum of a chroniccarrier of hepatitis BsAg which blood serum contains HBsAg associatedparticles of a size of 16 to 50 nm of which at least some are in therange of 30-50 nm and is substantially free of anti-HBs and containse-antigen and abundant Dane particles and which has no dectectableanti-e antibody, at least 1 percent of said particles beingcharacterized by the presence of free, uncombined and unprecipitatede-antigen; B. removing other proteinaceous matter from said serum suchthat the serum contains less that 10% of proteinaceous matter other thanthat associated with HBsAg or e-antigen; C. inactivating any virus inthe serum; and D. diluting the antigen containing serum with aphysiologically acceptable medium.
 2. A process according to claim 1wherein the resultant product is thereafter introduced into a human orchimpanzee in order to induce production of anti-HBs and anti-e as wellas immunity against subsequent exposure to hepatitis B virus.
 3. Amethod according to claim 1 wherein step B is performed by maintainingthe pH of the blood material in the range of 4.4 to 4.7; mixing saidmaterial, while its pH is being contained in said range, withapproximately 3.0 to 4.5 weight percent polyethylene glycol based on thetotal weight of the admixture, to produce a precipitate containing typeB hepatitis antigen containing HBsAg and e-antigen; separatelyrecovering said precipitate, adding a sufficient amount of water theretoto present intermediate free material having a type B hepatitis antigenconcentration the same or higher than in the original blood material;causing the pH of said intermediate material to be in the range ofapproximately 4.9 to 5.1 thereby producing a precipitate containing nonHB associated proteinaceous material, and polyethylene glycol, and afluid phase containing type B hepatitis/e -antigen particles; separatelyrecovering said fluid phase and adjusting the pH thereof to within therange of approximately 4.4 to 4.7; mixing the said fluid phase, whilemaintaining its pH within said range, with approximately 3.0 to 4.5weight percent polyethylene glycol based on the total weight of saidmixture, to produce a precipitate containing purified HBsAg/e particles;and recovering said purified HBsAg/e-containing material havingproteinaceous filamentous or Dane particles containing unprecipitatedfree, uncombined e-antigen.